2 downloads 6 Views 1MB Size Report
BY HELENE E ROSENBERG,' STEVEN J. ACKERMAN,'. AND DANIEL G. TENENT. From the Divisions of 'Infectious Diseases and lHematology/Oncology, ...

HUMAN EOSINOPHIL CATIONIC PROTEIN Molecular Cloning of a Cytotoxin and Helminthotoxin with Ribonuclease Activity BY HELENE E ROSENBERG,' STEVEN J . ACKERMAN,' AND DANIEL G . TENENT From the Divisions of 'Infectious Diseases and lHematology/Oncology, Department of Medicine, Harvard Medical School; and the Charles A . Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts 02215

The eosinophil cationic protein (ECP)' is one of several small, distinct argininerich proteins that have been isolated from the eosinophil's large specific granule. The molecular mass of human ECP has been estimated at 18-21 kD (1-3), depending on the degree of glycosylation (3). ECP has been shown to possess a wide variety of biological activities, including the ability to stimulate factor XII-dependent coagulation pathways (4), to neutralize the anticoagulant effects of heparin (5), and to inhibit lymphocyte proliferation induced by PHA or mixed lymphocyte reactions (6) . ECP is a potent cytotoxin; ECP-containing supernatants of activated eosinophils have been shown to be toxic to isolated myocardial cells in vitro (7), and both ECP and the eosinophil granule major basic protein (MBP) were found in the endothelial and endomyocardial lesions characteristic of the hypereosinophilic syndrome (8). Furthermore, ECP is also a potent helminthotoxin; destruction of schistosomula of Schistosoma mansont was reported at concentrations as low as 10' M (8-10-fold more active than MBP) (9, 10) . ECP has also been shown to kill trypomastigote and amastigote stages of Trypanosoma cruzi (11) . Both ECP and the related granule protein, eosinophil-derived neurotoxin (EDN), induce the neurotoxic effect known as the Gordon phenomenon (12) when injected into the cerebrospinal fluid of ex-

perimental animals (3, 13-15) . Although the molecular mechanism(s) by which ECP carries out these varied biologic activities is not clear, Young et al. (16) have suggested that ECP might damage cells by a colloid-osmotic process, as they have shown that it can introduce non-ion selective pores in both cellular and synthetic membranes . Gleich et al . (3) published the NH2-terminal sequences of ECP and the related granule protein, EDN ; the NH2-terminal sequence of ECP showed 67% identity to that of EDN, and 26% identity to the sequence of human pancreatic ribonuclease

This work was supported by National Institutes of Health grants AI-25230, AI-22660 (to S . J. Ackerman), CA-41456 (to D. G . Tenen), and IU41 RR01685 (to BIONET). D. G . Tenen is supported by a special fellowship from the Leukemia Society of America . Address correspondence to Helene F. Rosenberg, Department of Medicine, Division of Infectious Diseases, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215 . 1 Abbreviations used in this paper: ANG, angiogenin ; ECP, eosinophil cationic protein; EDN, eosinophilderived neurotoxin ; HLR, human liver ribonuclease ; HNSR, human nonsecretory ribonuclease ; HPR, human pancreatic ribonuclease ; MBP, major basic protein . J . Exp . MED. © The Rockefeller University Press - 0022-1007/89/07/0163/14 $2 .00 Volume 170 July 1989 163-176




(HPR), suggesting that ECP and EDN might belong to the ribonuclease multigene family. ECP and EDN were subsequently found to have ribonuclease activity, with ECP showing 100-fold less activity than EDN against a yeast RNA substrate (17, 18). We have previously isolated and sequenced a full-length cDNA clone for EDN (19); the complete cDNA-derived amino acid sequence was identical to a human nonsecretory ribonuclease isolated from urine (HNSR) (19, 20) . In addition, there was 32 % sequence identity between EDN and HPR, including conservation of the structural cysteine and catalytic lysine and histidine residues. We now report the isolation of a full-length cDNA clone for ECP The predicted amino acid sequence of mature ECP shows 66% sequence identity with EDN/HNSR, but has 12 more arginine residues and a higher net positive charge . While EDN is expressed in a wide variety of hematopoietic cells and cell lines (19), ECP appears to be expressed only in eosinophils and their precursors. Materials and Methods Peripheral blood granulocytes obtained by leukapheresis of a patient with the hypereosinophilic syndrome were purified as described (19). After two washes in cold HBSS and lysis ofRBC by brief suspension in cold lysis buffer (100 mM potassium carbonate, 150 mM ammonium chloride, and 0.1 mM EDTA, pH 7.2), the resuspended cell pellet was underlayered with a cushion of Ficoll-Hypaque (1.077 g/ml; Pharmacia Fine Chemicals, Piscataway, NJ) and centrifuged at 400 g for 30 min at room temperature. The mature granulocytes in the pellet were separated from cells suspended in the Ficoll-Hypaque layer (hypodense granulocytes and some mononuclear cells), and the RNA was extracted and purified by guanidium-isothiocyanate extraction followed by cesium chloride density gradient centrifugation (21). RNA from discarded samples ofnormal bone marrow cells was purified as described except without cell separation over Ficoll-Hypaque. Monocytes were purified from peripheral blood of normal donors by Ficoll-Hypaque density centrifugation and adherence to plastic . Neutrophil RNA was purified from peripheral blood neutrophils obtained from normal donors. Cells were washed and separated over FicollHypaque as above; in each case, the pellets contained

Suggest Documents